Autoimmunity
ISSN: 0891-6934 (Print) 1607-842X (Online) Journal homepage: http://www.tandfonline.com/loi/iaut20
Heritability analysis of IgG4 antibodies in autoimmune thyroid disease I. M. Outschoorn, M. V. Talor, C. L. Burek, W. H. Hoffman & N. R. Rose To cite this article: I. M. Outschoorn, M. V. Talor, C. L. Burek, W. H. Hoffman & N. R. Rose (2014) Heritability analysis of IgG4 antibodies in autoimmune thyroid disease, Autoimmunity, 47:5, 320-326, DOI: 10.3109/08916934.2014.897331 To link to this article: http://dx.doi.org/10.3109/08916934.2014.897331
Published online: 13 Mar 2014.
Submit your article to this journal
Article views: 95
View related articles
View Crossmark data
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iaut20 Download by: [Gothenburg University Library]
Date: 08 October 2017, At: 14:44
http://informahealthcare.com/aut ISSN: 0891-6934 (print), 1607-842X (electronic) Autoimmunity, 2014; 47(5): 320–326 ! 2014 Informa UK Ltd. DOI: 10.3109/08916934.2014.897331
ORIGINAL ARTICLE
Heritability analysis of IgG4 antibodies in autoimmune thyroid disease I. M. Outschoorn1, M. V. Talor2, C. L. Burek2, W. H. Hoffman3, and N. R. Rose2,4 Immune Response Unit (Immunology), Instituto de Salud Carlos III, Centro Nacional de Microbiologia, Majadahonda, Madrid, Spain, 2Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, MA, USA, 3Section of Pediatric Endocrinology, Georgia Regents University (formerly Georgia Health Sciences University), Augusta, GA, USA, and 4The W Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MA, USA
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
1
Abstract
Keywords
A study of IgG4 autoantibody levels in juvenile thyroid disease patients showed evidence of heritability using the ROMP screening method. These levels increased with time despite the fact that total IgG antibody decreased with time. Evidence of heritability was demonstrated only in patients with high titers of autoantibodies to both thyroglobulin (Tg) and thyroperoxidase (TPO) unlike family members who may show high titers of one or the other and be asymptomatic at the time of sampling. Since high and low IgG4 levels give different heritability plots, these findings may represent a more severe fibrotic form of thyroiditis with a distinct genetic background. Hence a simple predictive approach is offered by this screening tool for the disease in patients and family members which may be helpful in the future to identify IgG4-related thyroiditis early in the course of disease without the requirement for biopsy.
Autoimmunity genetics, autoimmune thyroid disease, heritability, IgG4 related thyroid disease, ROMP plots
Introduction Recent reports suggest that elevated levels of autoantibodies in the IgG4 subclass signify a distinct form of autoimmune disease characterized by infiltration of IgG4 positive plasmacytes, extensive fibrosis and a poor clinical response [1–3]. In Hashimoto’s thyroiditis, disproportionately elevated IgG4 autoantibodies to thyroid-specific antigens have been associated with disease characterized by a younger age of onset, rapid progress and more diffuse low echogenicity [4–6]. These findings may represent a more severe or fibrotic form of thyroiditis with a distinct genetic background. IgG4 was shown to play a role in other diseases as well [7–10]. Of particular importance is the issue of whether this disease can be recognized based on autoantibody studies at earlier ages and whether early treatment could be initiated to avoid the irreversible fibrotic process associated with this subtype of autoimmune thyroiditis [11,12]. A related issue for future studies is whether the IgG4-related disease (IgG4RD) in general is found in African American (AA) as well as European American (EA) patients at similar or different rates [13]. This study was performed in a unique population of families where one child, usually a daughter, developed
Correspondence: Ingrid Maive Outschoorn, PhD, Instituto de Salud Carlos III, Unidad de Respuesta Inmune (Area de Inmunologia), Centro Nacional de Microbiologia, Majadahonda, 28220, Spain. E-mail: [email protected]; [email protected]
History Received 17 September 2013 Revised 10 February 2014 Accepted 17 February 2014 Published online 13 March 2014
autoimmune thyroid disease, either Hashimoto’s thyroiditis/ chronic lymphocytic thyroiditis (HT/CLT) or Graves’ thyrotoxicosis (TT). Heritability studies had already been carried out on all these families [14,15]. The present report focuses on CLT patients. We were able to study the index case as well as their parents and siblings over time, recording the IgG4 levels of antibody specific to one of the two major thyroid antigens, thyroglobulin (Tg) and the total autoantibody levels to both Tg and thyroperoxidase (TPO). The primary goal of the study was to determine whether there is preliminary evidence of a heritable, unique subtype of autoimmune thyroid disease [16] or whether several genes make more modest contributions [17]. Because this was a retrospective study of stored serum samples, clinical records were not available to us. In order to estimate heritability in our population, we used a simple screening method, Regression of Offspring on Mid-Parent, or ROMP [18,19]. The data provided a preliminary estimate of the genetic contribution to a subtype of possible fibrotic disease within these families at a time when biopsy studies of fibrosis would not have been ethically acceptable. Some families participating in this study have been followed for up to 15 years after diagnosis and their subclass distributions over time offered further insights for future investigations. We were also able to include two ethnic groups (African American, AA or European American, EA) of juvenile patients along with their respective families diagnosed with thyrotoxicosis of Graves’ disease or hypothyroidism of CLT.
IgG4-related thyroid disease heritability
DOI: 10.3109/08916934.2014.897331
Materials and methods Population Thirty-four families previously studied [14] and seven control families of similar age range were included. At least one juvenile in each family was diagnosed as having CLT or TT. Chronic lymphocytic thyroiditis The population comprised of 18 juvenile patients, aged 7–19 years. One family had twin daughters, both probands. Total of 86 family members, all European American had up to 3 or 4 samples from each individual for assay. Titers ranged from a few mg/ml to a few hundred over the 15 years of study.
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
Graves’ thyrotoxicosis The 16 families with probands aged 6–23 years were included. Fifty-two family members came from nine European American families (TTw) and 37 family members from seven African American families (TTb), giving a total of 89 individuals. This group also included one family with twin daughters having high autoantibody titers to both autoantigens.
321
consistency, as well as on more than one occasion to control both for intra-assay and interassay variation. Requirements for heritability plots are as follows: (1) A group of patients with either parent or with an offspring sampled simultaneously. If samples from both parents and/or affected and unaffected siblings are available, further comparisons can be made. (2) A positive slope in this regression analysis allows one to consider this parameter as being heritable; i.e. it is worthwhile to examine other isotypes, both classes and subclasses, in these serum samples. Slopes may also differ when parental influences are compared. Maternal and paternal influences may give different slopes, suggesting maternal imprinting. (3) Samples could be grouped and re-examined on the basis of age and time since diagnosis. Patients with autoimmune thyroid disease (AITD) are usually treated and over the years their autoantibody titers decrease. Their unaffected siblings, moreover, may develop autoimmune disease over the course of the investigation. Hence, a positive heritability plot reflects parental/genetic influences better than such non-heritable ‘‘environmental’’ effects.
Thyroid autoantibodies
Results
Assays developed in our laboratory using a chromic chloride hemagglutination (HA) [15], for anti-TPO assays. Ranges varied from undetectable to titers of hundreds of thousands. Units for this assay were arbitrary ones correlating with the amount of chromium chloride dissociation. Subsequently total antibody titers and each of the IgG subclasses were measured by a quantitative ELISA [20]. Briefly units for this assay were micrograms/milliliter (mg/ml), using an in house standard curve calibrated with IUIS/WHO standards recommended and available at the time (cf. [21,22]).
Among the 18 juvenile CLT patients, seven of the probands attained a level of 20% of anti-Tg antibodies in the IgG4 subclass in at least one of the serum samples tested during the course of study. This was considered to be the high IgG4 group (Figure 1A and B). All other probands, the low IgG4 group, showed decreasing titers of IgG4 antibody to both Tg and TPO autoantigens over time, although occasionally the IgG4 titer showed some fluctuations. When analyzed by ROMP, IgG4 thyroid autoantibodies proved to be heritable in those patients who have high proportions of autoantibodies to Tg in the IgG4 subclass. In this subgroup of HT/CLT patients, although their total autoantibody titers to Tg were decreasing, the proportion of their anti-Tg antibodies in the IgG4 subclass increased in 4 of 7 patients. This IgG4 dominance changed over time (data not shown). Therefore, longitudinal studies are necessary, particularly in the early years following diagnosis. It is, therefore, possible that many of these patients could be considered to have IgG4-related diseases. In contrast, all CLT patients with low IgG4 levels gave negative heritability plots (Figure 2). Total anti-Tg concentrations ranged as high as 100 mg/ml for probands. High anti-TPO levels were found in both the high and the low IgG4 anti-Tg antibody groups. Maternal and paternal autoantibody levels thus did not correlate with high and low IgG4 in the affected children (data not shown). ROMP was not plotted for siblings since they usually had very low levels, by and large, less than 10 mg/ml. Maternal and paternal heritability plots of anti-Tg antibodies of the IgG4 subclass were compared (Figure 2B and E, and C and F, respectively). The number of parents participating in this longitudinal study over time totaled 22. These were mainly CLT families. Fathers gave more positive slopes but this did not reach statistical significance. The parents often
IgG subclass distribution patterns Subclass distributions reflected the percentages of each of the four IgG subclass concentrations in serum. Balanced patterns were reported when anti-Tg antibodies comprised between 20 and 30% of each of the four IgG subclasses. Some of the subclass assays used a mixture of monoclonal antibody reagents specific for human Fab and human Fc fragments in equal proportions. With the exception of IgG3 where the antihuman reagents are all directed to hinge region epitopes, this gave an increased sensitivity down to ng levels in some ELISA assays. IgG4 was defined as over-expressed in probands or family members when it made up 20% or more of the total anti-Tg antibody concentration at the time of sampling. Heritability plots Regression of offspring on Mid-parent (ROMP) heritability plots were derived by plotting the antibody levels of the index cases (probands) or siblings against the mean levels of antibodies from either or both parents [18,19]. Samples were collected from all family members at the same time and assayed simultaneously in duplicate or triplicate for
322
I. M. Outschoorn et al.
Autoimmunity, 2014; 47(5): 320–326
(B) 50 (A)
Patient 1 140
Patient 2
Patient 5
Patient 2
Patient 6
Patient 3
Patient 7
45
Patient 3
Patient 4
40
Patient 4
120
Patient 1
Patient 5 35
Patient 6
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
Patient 7 30 % IgG4
Total anti-Tg IgG (µg/ml)
100
80
25
20
60
15 40 10 20 5
0
0 0
2
4
6
8
10
Years post diagnosis
0
2
4
6
8
10
Years post diagnosis
Figure 1. The evolution of anti-Tg antibodies over the years of study of 7 high IgG4 titered HT/CLT juvenile patients (7 females) comparing the levels of total IgG autoantibodies (A) with the percentages of IgG4 over the same time period (B).
had very high autoantibody titers (in the range of hundreds of mg/ml), but these often decreased over the years. Only one mother and one sister showed increased TPO antibody titers over time. Five mothers and three fathers had proportions of IgG4 in the 20 to 50% range while one mother and one father had total anti-Tg concentrations of 200–450 mg/ml. Although these somewhat unusual individuals belonged to different families, only the former showed an increase in IgG4. Table 1 shows the results of total IgG and subclass distributions found in the high and low IgG4 subclass patient groups and their parents. No clear trend can be observed from these distributions. The most interesting feature of these studies, however, is the change over time. In Table 2, the change between the last sample with respect to the first measurement shows that for the high IgG4 patient group, while total IgG folds decreased, the relative fraction of IgG4 increased. In the case of the low IgG4 patient group, this relationship is not as clear. The patient numbers are smaller due to the reduced numbers of samples obtained over time for the entire families. In a family with twin daughters, both probands, as well as two of the three brothers, all had considerable titers of IgG4 autoantibody. One brother initially had his entire antibody response directed to Tg in the IgG4 subclass, but within a few years no longer had detectable IgG4 to Tg. The antibody profile to Tg changed to being entirely IgG1 and IgG2. The second brother with high IgG4 showed a decrease in this
subclass response, from 100% and from 46% IgG4 anti-Tg, while the third had a pattern of IgG2 dominance throughout the study. The mother initially had an unusual subclass restriction to only IgG3 (anti-Tg), although with very low titer. The father too had a low titer but no further samples were available for testing. In general the siblings with low IgG4 anti-Tg levels had high titers of IgG antibodies to TPO. In another family in this study, the proband’s husband had a balanced pattern of IgG subclasses in his antibodies to Tg. Their daughter had a high level of IgG4 like the grandmother but tended toward a balanced pattern by the time of the third sample 7 years later. Among the 19 siblings studied, the brothers and sisters of most of the CLT patients were asymptomatic for autoimmune thyroid disease although a few did have elevated TSH levels. In one family, patients from the high IgG4 group had a brother, and in the other family a brother and two sisters, with elevated TSH. In both families some siblings developed autoimmune thyroid disease subsequently. These siblings had autoantibodies to Tg although with lower levels than the probands. For purposes of comparison Figure 3 shows IgG1 heritability plots for the three groups of patients studied: CLT probands (Figure 3B) and GD/TT probands of two ethnic groups (European American and African American; Figure 3A and C, respectively). Such plots are less informative than the IgG4 heritability estimates that can be obtained on separating the patients on the basis of their high and low antiTg titers. Perhaps this is not as good a biomarker as IgG4,
IgG4-related thyroid disease heritability
DOI: 10.3109/08916934.2014.897331
(D) 2000
Probands IgG4 (ng/ml)
Probands IgG4 (ng/ml)
(A) y = 0.33x + 565 R2 = 0.05
1500 1000 500
1000
0 0
0
500
1000
1500
500
1500
2000 Probands IgG4 (ng/ml)
(E) 2000
y = 0.19x + 642 R2 = 0.023
1500 1000 500 0 0
500
1000
1500
1000
y = −0.36x + 495 R2 = 0.19
500
0
2000
0
500
Mothers IgG4 (ng/ml) (C)
(F) 2000
y = 0.26x + 644 R2 = 0.042
1500 1000 500 0 0
500
1000
1500
2000
1000 1500 Mothers IgG4 (ng/ml)
1000
Probands IgG4 (ng/ml)
Probands IgG4 (ng/ml)
1000
Mid-Parental Average IgG4 (ng/ml)
(B) Probands IgG4 (ng/ml)
y = −0.49x + 597 R2 = 0.46
500
0
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
323
2000
y = −0.24x + 493 R2 = 0.33
500
0 0
Fathers IgG4 (ng/ml)
500
1000
1500
2000
Fathers IgG4 (ng/ml)
Figure 2. Parental influences on two groups of HT/CLT patients separated on the basis of their high and low IgG4 levels using the method of ROMP plots to estimate heritability. In Figures 1(A and D), proband IgG4 anti-Tg autoantibody levels are plotted against the mid-parental averages assayed simultaneously while Figures 1(B–E) and C–F compare maternal and paternal effects separately (30). Table 1. Total IgG and subclass distributions for the high and low IgG4 patients. Distribution High Proband Mother Father Low Proband Mother Father
Total (mg/ml) Av. [range]
IgG1 (%) Av. [range]
IgG2 (%) Av. [range]
IgG3 (%) Av. [range]
IgG4 (%) Av. [range]
N
44 [7–138] 85 [2–456] 5 [1–17]
33 [19–49] 52 [13–85] 32 [18–57]
41 [20–56] 22 [0–50] 42 [10–76]
7 [2–16] 14 [0–65] 17 [1–36]
19 [1–48] 12 [0–34] 9 [1–23]
7 6 5
19 [2–55] 2 [1–2] 62 [1–186]
42 [18–126] 30 [10–50] 30 [17–48]
34 [21–65] 26 [10–45] 43 [23–70]
14 [2–34] 18 [9–32] 12 [1–28]
10 [5–17] 26 [9–54] 15 [6–33]
5 4 4
Table 2. Changes between last with respect to first measurements over time.
Fold High Proband Mother Father Low Proband Mother Father
Total (mg/ml) Av. [range]
IgG4 (%) Av. [range]
N
0.6 [0.0–1.3] 1.0 [0.3–2.6] 1.2 [0.5–2.3]
3.9 [0.5–21] 12.2 [0.1–67] 2.8 [0.3–10.7]
7 6 5
1.3 [1.0–3.3] 9.6 [1.0–33] 1.0 [0.1–1.9]
1.5 [0.4–5.4] 0.8 [0.0–1.7] 1.4 [0.5–3.2]
5 4 4
hence the interest in measuring IgG4 concentrations instead of total IgG or IgG1, which are often assayed routinely. In GD/TT patients IgG4 shows significant positive heritability (0.042) in the African American (AA) group. They differed from the European American (EA) group as in the African American patients both their IgG2 (0.004) and IgG3 (0.025) subclass plots showed significant positive heritability. For both the groups (AA and EA) the total anti-Tg autoantibodies showed positive heritability (0.001) when assayed by ELISA or by HA techniques. The EA probands also showed significant positive heritability for the IgG2 subclass (0.012). When the heritability plots for each IgG subclass were plotted separately, the GD/TT group did not
324
I. M. Outschoorn et al. 14 European American TT Patients y = 0.77x + 725 R2= 0,0061
(A) Probands IgG1 (ng/ml)
Autoimmunity, 2014; 47(5): 320–326
2000 1500 1000 500 0 0
500
1000
1500
2000
MidParentalAverageIgG1(ng/ml)
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
(B)
(C)
Figure 3. Probands IgG1 anti-Tg autoantibody levels plotted against mid-parental averages assayed simultaneously (100).
show a significantly positive slope for IgG1 (Figure 3). However, only the HT/CLT EA patients and family members could be followed over time. A difference in trend was observed here, an inversion of the slope, i.e. change from a positive regression to a negative one, between the parents but only in the case of IgG4. Therefore, in the AA patients with GD, parental influences differed more than in the two EA groups, particularly when IgG4 levels were compared.
Discussion The ROMP plot technique allows one to obtain a measurable difference between the maternal and paternal contributions to heritability whether they are examined separately for different ethnic groups or on the basis of high or low IgG4 anti-Tg autoantibodies. The most striking finding in this retrospective analysis study was that half of the juvenile thyroiditis patients, (most of whom were female), reached the level of 20% IgG4 at some point of the study period. This large proportion may become an early criterion for potential IgG4-related thyroid disease. In order to determine whether this condition was hereditary, we employed ROMP, a simple preliminary genetic assay. Using ROMP, IgG4 showed preliminary evidence of heritability in both the two major autoimmune thyroid diseases, TT and CLT. Our threshold for including a thyroiditis patient in the IgG4-related disease group was an IgG4 subclass of
approximately 20% of the total antibody. This may be particularly important in non-Japanese patients in line with efforts to tailor criteria to different ethnic origins among patients with other autoimmune diseases [23–27]. Since the various research algorithms available require larger patient numbers, ROMP followed by isotype distribution pattern analysis could yield further useful insight. Although we recognize that environmental influences also exert considerable effects on the development of autoimmune thyroid disease [28–31], the measure of IgG4 may lead to predictions for selecting patients most suited to B-cell therapeutic strategies [3,32,33]. As reported previously that detectable levels of one of the two autoantibodies (anti Tg and TPO) were often present in either parent or in one or more sibs. In contrast, both autoantibodies were usually high only in the clinically affected probands. Since the responses to the two main autoantigens seem to be independent, we examined the probands with high and low IgG4 levels and that of the siblings separately. CLT patients showed a significantly positive slope in the group with high IgG4 anti-Tg titers. This suggests that autoantibody levels in the IgG4 subclass may become a marker for a heritable disease phenotype in CLT patients. In contrast, probands with low IgG4 titers gave negative heritability plots. However, IgG4 dominance can change over time and underscores the importance of repeated testing. The appearance of a positive slope may lead to the problem of the predictability of this analysis, in particular, when one or the other parent presents with a high IgG4 autoantibody titer. The number of siblings in this study was small and they had very low levels. Attempts have been made to address such problems using family-based study designs, in order to study genetic associations with large effects. However, next-generation sequencing technologies such as GWAS are extremely costly. ROMP (a cheap method) offers the advantage of not requiring parental genotypes [34,35]. Perhaps with a larger cohort of patients, such ROMP plots for age cohorts may prove feasible [36]. Previous work on the subclass distributions of autoantibodies in autoimmune thyroid disease described relative restriction to the IgG1 and IgG4 subclasses [37,38]. Perhaps this reflects a selected group of patients with high autoantibody titers or a particularly severe presentation, as seen in one of the mothers in our own study population. More varied distribution patterns among both adult and juvenile patients were observed when more sensitive assays were used [20]. Further studies on biopsies may enable the determination as to whether IgG4 elevation may be used as an early indicator of fibrotic disease. This may also facilitate the selection of family members among those screened for autoantibodies for follow-up in order to diagnose them early and initiate treatment if necessary. Perhaps early intervention could aid in preventing the progression to fibrosis [7,11], and unresponsiveness to steroid therapy, as has been shown for Mickulicz Disease patients [39]. In centers where a wider availability of family members may allow more of them to participate in studies, either at early stages of disease before treatment is initiated, a selection could be made that may
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
DOI: 10.3109/08916934.2014.897331
enable siblings to be screened over the years for early development of autoimmune diseases. This is particularly important when more than one autoantibody titer is increasing over time [40]. With respect to the siblings studies here with two autoantibodies that differ with respect to parents, the presence of more than one autoantibody could be considered as an early warning sign of autoimmune disease but not necessarily of a fibrotic variant [41–44]. In recent years several studies on autoimmune thyroid disease have been published with regard to various autoimmune diseases and ethnic groups [45–50]. Among these several are concerned with risk factors affecting susceptibility to GD/TT. An extensive comparison of susceptibility studies of both autoimmune thyroid diseases and of HT/CLT alone concludes that due to these ethnic differences, gene-gene and gene-environment interactions may be a more useful approach to study contributing or predisposing genes [17,51,52]. Renewed recognition of the role of antibodies to Tg, genetic linkage to HLA [53] and the skewed chromosomal inactivation to explain female preponderance [54,55] have aided interdisciplinary research in this area. In conclusion we suggest that screening may be able to differentiate these two subgroups of CLT patients by this simple method and allow those patients with IgG4-related thyroid disease to be treated earlier.
Declaration of interest The authors declare no conflicts of interest.
References 1. Masaki, Y., N. Kurose, and H. Umehara. 2011. IgG4-related disease: a novel lymphoproliferative disorder discovered and established in Japan in the 21st century. J. Clin. Exp. Hematop. 51: 13–20. 2. Ebbo, M., A. Grados, E. Bernit, et al. 2012. Pathologies associated with serum IgG4 elevation. Int. J. Rheumatol. 2012: 602809. 3. Mahajan, V. S., H. Mattoo, V. Deshpande, et al. 2014. IgG4-Related Disease. Annu. Rev. Pathol. Mech. Dis. 9: 315–347. 4. Li, Y., Y. Bai, and Z. Liu, et al. 2009. Immunohistochemistry of IgG4 can help subclassify Hashimoto’s autoimmune thyroiditis. Pathol. Int. 59: 636–641. 5. Zhang, J., L. Zhao, Y. Gao, et al. 2013. A classification of Hashimoto’s thyroiditis based on immunohistochemistry for IgG4 and IgG. Thyroid. 24: 364–370. 6. Deshpande, V., A. Huck, E. Ooi, et al. 2012. Fibrosing variant of Hashimoto thyroiditis is an IgG4 related disease. J. Clin. Pathol. 65: 725–728. 7. Shimizu, Y., M. Yamamoto, Y. Naishiro, et al. 2013. Necessity of early intervention for IgG4-related disease-delayed treatment induces fibrosis progression. Rheumatology (Oxford). 52: 679–683. 8. Guma, M., and G. S. Firestein. 2012. IgG4-related diseases. Best. Pract. Res. Clin. Rheumatol. 26: 425–438. 9. Palazzo, E., C. Palazzo, and M. Palazzo. 2013. IgG4-related disease. Joint Bone Spine. 81: 27–31. 10. Kamisawa, T., S. T. Chari, M. M. Lerch, et al. 2014. Recent advances in autoimmune pancreatitis: type 1 and type 2. Postgrad. Med. J. 90: 18–25. 11. Koike, T. 2013. IgG4-related disease: why high IgG4 and fibrosis? Arthritis Res. Ther. 15: 103. 12. Lee, L. H., E. A. Shaffer, V. Falck, and M. M. Kelly. 2013. Solitary lesions with fibrosis and increased IgG4+ plasma cells: Part of the expanding spectrum of IgG4-related disease or a nonspecific inflammatory response? Int. J. Surg. Pathol. [Epub ahead of print]. doi: 10.1177/1066896913513832.
IgG4-related thyroid disease heritability
325
13. Moroni, L., I. Bianchi, and A. Lleo. 2012. Geoepidemiology, gender and autoimmune disease. Autoimmun. Rev. 11: A386–92. 14. Burek, C. L., W. H. Hoffman, and N. R. Rose. 1982. The presence of thyroid autoantibodies in children and adolescents with autoimmune thyroid disease and in their siblings and parents. Clin. Immunol. Immunopathol. 25: 395–404. 15. Burek, C.L., N. R. Rose, G. M. Najar, et al. 1984. Autoimmune Thyroid Disease. In Clinical Immunology, Vol. 1 – Immunogenetics. C. S. David, and G. S. Panayi, eds. Butterworths, London. p. 207–233. 16. Li, Y., E. Nishihara, and K. Kakudo. 2011. Hashimoto’s thyroiditis: old concepts and new insights. Curr. Opin. Rheumatol. 23: 102–107. 17. Jabrocka-Hybel, A., A. Skalniak, J. Pia˛tkowski, et al. 2013. How far are we from understanding the genetic basis of Hashimoto’s thyroiditis? Int. Rev. Immunol. 32: 337–354. 18. Manolio, T. A., K. C. Barnes, T. H. Beaty, et al. 2003. Sex differences in heritability of sensitization to Blomia tropicalis in asthma using regression of offspring on midparent (ROMP) methods. Hum. Genet. 113: 437–446. 19. Outschoorn, I. M., M. V. Talor, W. H. Hoffman, et al. 2011. Autoantibody heritability in thyroiditis: IgG subclass contributions. Autoimmunity. 44: 195–200. 20. Kuppers, R. C., I. M. Outschoorn, R. G. Hamilton, et al. 1993. Quantitative measurement of human thyroglobulin-specific antibodies by use of a sensitive enzyme-linked immunoassay. Clin. Immunol. Immunopathol. 67: 68–77. 21. Beastall, G. H. 2014. What is the significance of antithyroid antibodies in children and adolescents? Clin. Biochem. 47: 1–2. 22. Taubner, K., G. Schubert, F. Pulzer, et al. 2014. Serum concentrations of anti-thyroid peroxidase and anti-thyroglobulin antibodies in children and adolescents without apparent thyroid disorders. Clin. Biochem. 47: 3–7. 23. Hart, P. A., T. Kamisawa, W. R. Brugge, et al. 2013. Long-term outcomes of autoimmune pancreatitis: a multicentre, international analysis. Gut. 62: 1771–1776. 24. Yamamoto, M., T. Tabeya, Y. Naishiro, et al. 2012. Value of serum IgG4 in the diagnosis of IgG4-related disease and in differentiation from rheumatic diseases and other diseases. Mod. Rheumatol. 22: 419–425. 25. Tsuboi, H., H. Asashima, C. Takai, et al. 2013. Primary and secondary surveys on epidemiology of Sjo¨gren’s syndrome in Japan. Mod. Rheumatol. [Epub ahead of print]. doi: 10.3109/ 14397595.2013.843765. 26. Yang, Y., X. Su, K. Zhang, and R. Zhou. 2013. The Fc receptor-like 3 gene polymorphisms and susceptibility to autoimmune diseases: an updated meta-analysis. Autoimmunity. 46: 547–558. 27. Yang, Y., K. Zhang, and R. Zhou. 2014. Meta-analysis of premiRNA polymorphisms association with susceptibility to autoimmune diseases. Immunol. Invest. 43: 13–27. 28. Duntas, L. H. 2011. Environmental factors and thyroid autoimmunity. Ann. Endocrinol. (Paris). 72: 108–113. 29. McLeod, D. S., and D. S. Cooper. 2012. The incidence and prevalence of thyroid autoimmunity. Endocrine. 42: 252–265. 30. Temajo, N. O., and N. Howard. 2014. The mosaic of environment involvement in autoimmunity: the abrogation of viral latency by stress, a non-infectious environmental agent, is an intrinsic prerequisite prelude before viruses can rank as infectious environmental agents that trigger autoimmune diseases. Autoimmun. Rev. [Epub ahead of print]. doi: 10.1016/j.autrev.2013.12.003. 31. de Buy Wenniger, L. J., E. L. Culver, and U. Beuers. 2014. Exposure to occupational antigens might predispose to IgG4related disease. Hepatology. [Epub ahead of print]. doi: 10.1002/ hep.26999. 32. Yamamoto, M., H. Takahashi, and K. Ishigami, et al. 2012. Relapse patterns in IgG4-related disease. Ann. Rheum. Dis. 71: 1755. 33. Murakami J., Matsui S., Ishizawa S., et al. 2013. Recurrence of IgG4-related disease following treatment with rituximab. Mod. Rheumatol. 23: 1226–1230. 34. Arlt, M. F., T. E. Wilson, and T. W. Glover. 2012. Replication stress and mechanisms of CNV formation. Curr. Opin. Genet. Dev. 22: 204–210.
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
326
I. M. Outschoorn et al.
35. Inoue, N., M. Watanabe, H. Yamada, et al. 2012. Associations between autoimmune thyroid disease prognosis and functional polymorphisms of susceptibility genes, CTLA4, PTPN22, CD40, FCRL3, and ZFAT, previously revealed in genome-wide association studies. J. Clin. Immunol. 32: 1243–1252. 36. Brix, T. H., and L. Hegedu¨s. 2012. Twin studies as a model for exploring the aetiology of autoimmune thyroid disease. Clin. Endocrinol (Oxf). 76: 457–464. 37. Parkes, A. B., S. M. McLachlan, P. Bird, and B. Rees Smith. 1984. The distribution of microsomal and thyroglobulin antibody activity among the IgG subclasses. Clin. Exp. Immunol. 57: 239–243. 38. McLachlan, S. M., and B. Rapoport. 2008. Thyroid peroxidase autoantibody epitopes revisited*. Clin. Endocrinol (Oxf). 69: 526–527. 39. Yamamoto, M., H. Takahashi, K. Ishigami, et al. 2012. Evaluation and clinical validity of a new questionnaire for Mikulicz’s disease. Int. J. Rheumatol. 2012: 283459. 40. Watanabe, T., M. Maruyama, T. Ito, et al. 2013. Two siblings with type 1 autoimmune pancreatitis. Intern. Med. 52: 895–899. 41. Okazaki, K., and H. Umehara. 2012. Are Classification Criteria for IgG4-RD Now Possible? The concept of IgG4-related disease and proposal of comprehensive diagnostic criteria in Japan. Int. J. Rheumatol. 2012: 357071. 42. Watanabe, T., M. Maruyama, T. Ito, et al. 2013. Clinical features of a new disease concept, IgG4-related thyroiditis. Scand. J. Rheumatol. 42: 325–330. 43. Kawashima, S. T., T. Tagami, K. Nakao, et al. 2013. Serum levels of IgG and IgG4 in Hashimoto thyroiditis. Endocrine. 45: 236–243. 44. Li, D., W. Cai, R. Gu, et al. 2013. Th17 cell plays a role in the pathogenesis of Hashimoto’s thyroiditis in patients. Clin. Immunol. 149: 411–420.
Autoimmunity, 2014; 47(5): 320–326
45. Ban, Y., T. Tozaki, M. Taniyama, et al. 2012. Multiple SNPs in intron 41 of thyroglobulin gene are associated with autoimmune thyroid disease in the Japanese population. PLoS One. 7: e37501. 46. Ban, Y., T. Tozaki, and M. Taniyama. 2013. The replication of the association of the rs9355610 within 6p27 with Graves’ disease. Autoimmunity. 46: 395–398. 47. Alkhateeb, A., N. A. Marzouka, and R. Tashtoush. 2013. Variants in PTPN22 and SMOC2 genes and the risk of thyroid disease in the Jordanian Arab population. Endocrine. 44: 702–709. 48. Alkhateeb, A., Y. Jarun, and R. Tashtoush. 2013. Polymorphisms in NLRP1 gene and susceptibility to autoimmune thyroid disease. Autoimmunity. 46: 215–221. 49. Zaaber, I., S. Mestiri, H. Marmouch, et al. 2013. Polymorphisms in TSHR and IL1RN genes and the risk and prognosis of Hashimoto’s thyroiditis. Autoimmunity. 47: 113–118. 50. Yan, N., Y. L. Yu, J. Yang, et al. 2012. Association of interleukin17A and -17F gene single-nucleotide polymorphisms with autoimmune thyroid diseases. Autoimmunity. 45: 533–539. 51. Weetman, A. 2013. A hundred years of Hashimoto’s thyroiditis. Thyroid. 23: 135–136. 52. Culver, E. L., and A. C. Bateman. 2013. General principles of IgG4-related disease. Mini-Symposium. Diagnostic Histopathology. 19:111–118. 53. Duntas, L.H., Y. Hiromatsu, and N. Amino. 2013. Centennial of the description of Hashimoto’s thyroiditis: two thought-provoking events. Thyroid. 23: 643–645. 54. Simmonds, M. J., F. K. Kavvoura, O. J. Brand, et al. 2014. Skewed x chromosome inactivation and female preponderance in autoimmune thyroid disease: an association study and meta-analysis. J. Clin. Endocrinol. Metab. 99: E127–E131. 55. Hunt, K. A., V. Mistry, N. A. Bockett, et al. 2013. Negligible impact of rare autoimmune-locus coding-region variants on missing heritability. Nature. 498: 232–235.
ISSN: 0891-6934 (Print) 1607-842X (Online) Journal homepage: http://www.tandfonline.com/loi/iaut20
Heritability analysis of IgG4 antibodies in autoimmune thyroid disease I. M. Outschoorn, M. V. Talor, C. L. Burek, W. H. Hoffman & N. R. Rose To cite this article: I. M. Outschoorn, M. V. Talor, C. L. Burek, W. H. Hoffman & N. R. Rose (2014) Heritability analysis of IgG4 antibodies in autoimmune thyroid disease, Autoimmunity, 47:5, 320-326, DOI: 10.3109/08916934.2014.897331 To link to this article: http://dx.doi.org/10.3109/08916934.2014.897331
Published online: 13 Mar 2014.
Submit your article to this journal
Article views: 95
View related articles
View Crossmark data
Citing articles: 2 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iaut20 Download by: [Gothenburg University Library]
Date: 08 October 2017, At: 14:44
http://informahealthcare.com/aut ISSN: 0891-6934 (print), 1607-842X (electronic) Autoimmunity, 2014; 47(5): 320–326 ! 2014 Informa UK Ltd. DOI: 10.3109/08916934.2014.897331
ORIGINAL ARTICLE
Heritability analysis of IgG4 antibodies in autoimmune thyroid disease I. M. Outschoorn1, M. V. Talor2, C. L. Burek2, W. H. Hoffman3, and N. R. Rose2,4 Immune Response Unit (Immunology), Instituto de Salud Carlos III, Centro Nacional de Microbiologia, Majadahonda, Madrid, Spain, 2Department of Pathology, The Johns Hopkins School of Medicine, Baltimore, MA, USA, 3Section of Pediatric Endocrinology, Georgia Regents University (formerly Georgia Health Sciences University), Augusta, GA, USA, and 4The W Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, MA, USA
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
1
Abstract
Keywords
A study of IgG4 autoantibody levels in juvenile thyroid disease patients showed evidence of heritability using the ROMP screening method. These levels increased with time despite the fact that total IgG antibody decreased with time. Evidence of heritability was demonstrated only in patients with high titers of autoantibodies to both thyroglobulin (Tg) and thyroperoxidase (TPO) unlike family members who may show high titers of one or the other and be asymptomatic at the time of sampling. Since high and low IgG4 levels give different heritability plots, these findings may represent a more severe fibrotic form of thyroiditis with a distinct genetic background. Hence a simple predictive approach is offered by this screening tool for the disease in patients and family members which may be helpful in the future to identify IgG4-related thyroiditis early in the course of disease without the requirement for biopsy.
Autoimmunity genetics, autoimmune thyroid disease, heritability, IgG4 related thyroid disease, ROMP plots
Introduction Recent reports suggest that elevated levels of autoantibodies in the IgG4 subclass signify a distinct form of autoimmune disease characterized by infiltration of IgG4 positive plasmacytes, extensive fibrosis and a poor clinical response [1–3]. In Hashimoto’s thyroiditis, disproportionately elevated IgG4 autoantibodies to thyroid-specific antigens have been associated with disease characterized by a younger age of onset, rapid progress and more diffuse low echogenicity [4–6]. These findings may represent a more severe or fibrotic form of thyroiditis with a distinct genetic background. IgG4 was shown to play a role in other diseases as well [7–10]. Of particular importance is the issue of whether this disease can be recognized based on autoantibody studies at earlier ages and whether early treatment could be initiated to avoid the irreversible fibrotic process associated with this subtype of autoimmune thyroiditis [11,12]. A related issue for future studies is whether the IgG4-related disease (IgG4RD) in general is found in African American (AA) as well as European American (EA) patients at similar or different rates [13]. This study was performed in a unique population of families where one child, usually a daughter, developed
Correspondence: Ingrid Maive Outschoorn, PhD, Instituto de Salud Carlos III, Unidad de Respuesta Inmune (Area de Inmunologia), Centro Nacional de Microbiologia, Majadahonda, 28220, Spain. E-mail: [email protected]; [email protected]
History Received 17 September 2013 Revised 10 February 2014 Accepted 17 February 2014 Published online 13 March 2014
autoimmune thyroid disease, either Hashimoto’s thyroiditis/ chronic lymphocytic thyroiditis (HT/CLT) or Graves’ thyrotoxicosis (TT). Heritability studies had already been carried out on all these families [14,15]. The present report focuses on CLT patients. We were able to study the index case as well as their parents and siblings over time, recording the IgG4 levels of antibody specific to one of the two major thyroid antigens, thyroglobulin (Tg) and the total autoantibody levels to both Tg and thyroperoxidase (TPO). The primary goal of the study was to determine whether there is preliminary evidence of a heritable, unique subtype of autoimmune thyroid disease [16] or whether several genes make more modest contributions [17]. Because this was a retrospective study of stored serum samples, clinical records were not available to us. In order to estimate heritability in our population, we used a simple screening method, Regression of Offspring on Mid-Parent, or ROMP [18,19]. The data provided a preliminary estimate of the genetic contribution to a subtype of possible fibrotic disease within these families at a time when biopsy studies of fibrosis would not have been ethically acceptable. Some families participating in this study have been followed for up to 15 years after diagnosis and their subclass distributions over time offered further insights for future investigations. We were also able to include two ethnic groups (African American, AA or European American, EA) of juvenile patients along with their respective families diagnosed with thyrotoxicosis of Graves’ disease or hypothyroidism of CLT.
IgG4-related thyroid disease heritability
DOI: 10.3109/08916934.2014.897331
Materials and methods Population Thirty-four families previously studied [14] and seven control families of similar age range were included. At least one juvenile in each family was diagnosed as having CLT or TT. Chronic lymphocytic thyroiditis The population comprised of 18 juvenile patients, aged 7–19 years. One family had twin daughters, both probands. Total of 86 family members, all European American had up to 3 or 4 samples from each individual for assay. Titers ranged from a few mg/ml to a few hundred over the 15 years of study.
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
Graves’ thyrotoxicosis The 16 families with probands aged 6–23 years were included. Fifty-two family members came from nine European American families (TTw) and 37 family members from seven African American families (TTb), giving a total of 89 individuals. This group also included one family with twin daughters having high autoantibody titers to both autoantigens.
321
consistency, as well as on more than one occasion to control both for intra-assay and interassay variation. Requirements for heritability plots are as follows: (1) A group of patients with either parent or with an offspring sampled simultaneously. If samples from both parents and/or affected and unaffected siblings are available, further comparisons can be made. (2) A positive slope in this regression analysis allows one to consider this parameter as being heritable; i.e. it is worthwhile to examine other isotypes, both classes and subclasses, in these serum samples. Slopes may also differ when parental influences are compared. Maternal and paternal influences may give different slopes, suggesting maternal imprinting. (3) Samples could be grouped and re-examined on the basis of age and time since diagnosis. Patients with autoimmune thyroid disease (AITD) are usually treated and over the years their autoantibody titers decrease. Their unaffected siblings, moreover, may develop autoimmune disease over the course of the investigation. Hence, a positive heritability plot reflects parental/genetic influences better than such non-heritable ‘‘environmental’’ effects.
Thyroid autoantibodies
Results
Assays developed in our laboratory using a chromic chloride hemagglutination (HA) [15], for anti-TPO assays. Ranges varied from undetectable to titers of hundreds of thousands. Units for this assay were arbitrary ones correlating with the amount of chromium chloride dissociation. Subsequently total antibody titers and each of the IgG subclasses were measured by a quantitative ELISA [20]. Briefly units for this assay were micrograms/milliliter (mg/ml), using an in house standard curve calibrated with IUIS/WHO standards recommended and available at the time (cf. [21,22]).
Among the 18 juvenile CLT patients, seven of the probands attained a level of 20% of anti-Tg antibodies in the IgG4 subclass in at least one of the serum samples tested during the course of study. This was considered to be the high IgG4 group (Figure 1A and B). All other probands, the low IgG4 group, showed decreasing titers of IgG4 antibody to both Tg and TPO autoantigens over time, although occasionally the IgG4 titer showed some fluctuations. When analyzed by ROMP, IgG4 thyroid autoantibodies proved to be heritable in those patients who have high proportions of autoantibodies to Tg in the IgG4 subclass. In this subgroup of HT/CLT patients, although their total autoantibody titers to Tg were decreasing, the proportion of their anti-Tg antibodies in the IgG4 subclass increased in 4 of 7 patients. This IgG4 dominance changed over time (data not shown). Therefore, longitudinal studies are necessary, particularly in the early years following diagnosis. It is, therefore, possible that many of these patients could be considered to have IgG4-related diseases. In contrast, all CLT patients with low IgG4 levels gave negative heritability plots (Figure 2). Total anti-Tg concentrations ranged as high as 100 mg/ml for probands. High anti-TPO levels were found in both the high and the low IgG4 anti-Tg antibody groups. Maternal and paternal autoantibody levels thus did not correlate with high and low IgG4 in the affected children (data not shown). ROMP was not plotted for siblings since they usually had very low levels, by and large, less than 10 mg/ml. Maternal and paternal heritability plots of anti-Tg antibodies of the IgG4 subclass were compared (Figure 2B and E, and C and F, respectively). The number of parents participating in this longitudinal study over time totaled 22. These were mainly CLT families. Fathers gave more positive slopes but this did not reach statistical significance. The parents often
IgG subclass distribution patterns Subclass distributions reflected the percentages of each of the four IgG subclass concentrations in serum. Balanced patterns were reported when anti-Tg antibodies comprised between 20 and 30% of each of the four IgG subclasses. Some of the subclass assays used a mixture of monoclonal antibody reagents specific for human Fab and human Fc fragments in equal proportions. With the exception of IgG3 where the antihuman reagents are all directed to hinge region epitopes, this gave an increased sensitivity down to ng levels in some ELISA assays. IgG4 was defined as over-expressed in probands or family members when it made up 20% or more of the total anti-Tg antibody concentration at the time of sampling. Heritability plots Regression of offspring on Mid-parent (ROMP) heritability plots were derived by plotting the antibody levels of the index cases (probands) or siblings against the mean levels of antibodies from either or both parents [18,19]. Samples were collected from all family members at the same time and assayed simultaneously in duplicate or triplicate for
322
I. M. Outschoorn et al.
Autoimmunity, 2014; 47(5): 320–326
(B) 50 (A)
Patient 1 140
Patient 2
Patient 5
Patient 2
Patient 6
Patient 3
Patient 7
45
Patient 3
Patient 4
40
Patient 4
120
Patient 1
Patient 5 35
Patient 6
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
Patient 7 30 % IgG4
Total anti-Tg IgG (µg/ml)
100
80
25
20
60
15 40 10 20 5
0
0 0
2
4
6
8
10
Years post diagnosis
0
2
4
6
8
10
Years post diagnosis
Figure 1. The evolution of anti-Tg antibodies over the years of study of 7 high IgG4 titered HT/CLT juvenile patients (7 females) comparing the levels of total IgG autoantibodies (A) with the percentages of IgG4 over the same time period (B).
had very high autoantibody titers (in the range of hundreds of mg/ml), but these often decreased over the years. Only one mother and one sister showed increased TPO antibody titers over time. Five mothers and three fathers had proportions of IgG4 in the 20 to 50% range while one mother and one father had total anti-Tg concentrations of 200–450 mg/ml. Although these somewhat unusual individuals belonged to different families, only the former showed an increase in IgG4. Table 1 shows the results of total IgG and subclass distributions found in the high and low IgG4 subclass patient groups and their parents. No clear trend can be observed from these distributions. The most interesting feature of these studies, however, is the change over time. In Table 2, the change between the last sample with respect to the first measurement shows that for the high IgG4 patient group, while total IgG folds decreased, the relative fraction of IgG4 increased. In the case of the low IgG4 patient group, this relationship is not as clear. The patient numbers are smaller due to the reduced numbers of samples obtained over time for the entire families. In a family with twin daughters, both probands, as well as two of the three brothers, all had considerable titers of IgG4 autoantibody. One brother initially had his entire antibody response directed to Tg in the IgG4 subclass, but within a few years no longer had detectable IgG4 to Tg. The antibody profile to Tg changed to being entirely IgG1 and IgG2. The second brother with high IgG4 showed a decrease in this
subclass response, from 100% and from 46% IgG4 anti-Tg, while the third had a pattern of IgG2 dominance throughout the study. The mother initially had an unusual subclass restriction to only IgG3 (anti-Tg), although with very low titer. The father too had a low titer but no further samples were available for testing. In general the siblings with low IgG4 anti-Tg levels had high titers of IgG antibodies to TPO. In another family in this study, the proband’s husband had a balanced pattern of IgG subclasses in his antibodies to Tg. Their daughter had a high level of IgG4 like the grandmother but tended toward a balanced pattern by the time of the third sample 7 years later. Among the 19 siblings studied, the brothers and sisters of most of the CLT patients were asymptomatic for autoimmune thyroid disease although a few did have elevated TSH levels. In one family, patients from the high IgG4 group had a brother, and in the other family a brother and two sisters, with elevated TSH. In both families some siblings developed autoimmune thyroid disease subsequently. These siblings had autoantibodies to Tg although with lower levels than the probands. For purposes of comparison Figure 3 shows IgG1 heritability plots for the three groups of patients studied: CLT probands (Figure 3B) and GD/TT probands of two ethnic groups (European American and African American; Figure 3A and C, respectively). Such plots are less informative than the IgG4 heritability estimates that can be obtained on separating the patients on the basis of their high and low antiTg titers. Perhaps this is not as good a biomarker as IgG4,
IgG4-related thyroid disease heritability
DOI: 10.3109/08916934.2014.897331
(D) 2000
Probands IgG4 (ng/ml)
Probands IgG4 (ng/ml)
(A) y = 0.33x + 565 R2 = 0.05
1500 1000 500
1000
0 0
0
500
1000
1500
500
1500
2000 Probands IgG4 (ng/ml)
(E) 2000
y = 0.19x + 642 R2 = 0.023
1500 1000 500 0 0
500
1000
1500
1000
y = −0.36x + 495 R2 = 0.19
500
0
2000
0
500
Mothers IgG4 (ng/ml) (C)
(F) 2000
y = 0.26x + 644 R2 = 0.042
1500 1000 500 0 0
500
1000
1500
2000
1000 1500 Mothers IgG4 (ng/ml)
1000
Probands IgG4 (ng/ml)
Probands IgG4 (ng/ml)
1000
Mid-Parental Average IgG4 (ng/ml)
(B) Probands IgG4 (ng/ml)
y = −0.49x + 597 R2 = 0.46
500
0
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
323
2000
y = −0.24x + 493 R2 = 0.33
500
0 0
Fathers IgG4 (ng/ml)
500
1000
1500
2000
Fathers IgG4 (ng/ml)
Figure 2. Parental influences on two groups of HT/CLT patients separated on the basis of their high and low IgG4 levels using the method of ROMP plots to estimate heritability. In Figures 1(A and D), proband IgG4 anti-Tg autoantibody levels are plotted against the mid-parental averages assayed simultaneously while Figures 1(B–E) and C–F compare maternal and paternal effects separately (30). Table 1. Total IgG and subclass distributions for the high and low IgG4 patients. Distribution High Proband Mother Father Low Proband Mother Father
Total (mg/ml) Av. [range]
IgG1 (%) Av. [range]
IgG2 (%) Av. [range]
IgG3 (%) Av. [range]
IgG4 (%) Av. [range]
N
44 [7–138] 85 [2–456] 5 [1–17]
33 [19–49] 52 [13–85] 32 [18–57]
41 [20–56] 22 [0–50] 42 [10–76]
7 [2–16] 14 [0–65] 17 [1–36]
19 [1–48] 12 [0–34] 9 [1–23]
7 6 5
19 [2–55] 2 [1–2] 62 [1–186]
42 [18–126] 30 [10–50] 30 [17–48]
34 [21–65] 26 [10–45] 43 [23–70]
14 [2–34] 18 [9–32] 12 [1–28]
10 [5–17] 26 [9–54] 15 [6–33]
5 4 4
Table 2. Changes between last with respect to first measurements over time.
Fold High Proband Mother Father Low Proband Mother Father
Total (mg/ml) Av. [range]
IgG4 (%) Av. [range]
N
0.6 [0.0–1.3] 1.0 [0.3–2.6] 1.2 [0.5–2.3]
3.9 [0.5–21] 12.2 [0.1–67] 2.8 [0.3–10.7]
7 6 5
1.3 [1.0–3.3] 9.6 [1.0–33] 1.0 [0.1–1.9]
1.5 [0.4–5.4] 0.8 [0.0–1.7] 1.4 [0.5–3.2]
5 4 4
hence the interest in measuring IgG4 concentrations instead of total IgG or IgG1, which are often assayed routinely. In GD/TT patients IgG4 shows significant positive heritability (0.042) in the African American (AA) group. They differed from the European American (EA) group as in the African American patients both their IgG2 (0.004) and IgG3 (0.025) subclass plots showed significant positive heritability. For both the groups (AA and EA) the total anti-Tg autoantibodies showed positive heritability (0.001) when assayed by ELISA or by HA techniques. The EA probands also showed significant positive heritability for the IgG2 subclass (0.012). When the heritability plots for each IgG subclass were plotted separately, the GD/TT group did not
324
I. M. Outschoorn et al. 14 European American TT Patients y = 0.77x + 725 R2= 0,0061
(A) Probands IgG1 (ng/ml)
Autoimmunity, 2014; 47(5): 320–326
2000 1500 1000 500 0 0
500
1000
1500
2000
MidParentalAverageIgG1(ng/ml)
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
(B)
(C)
Figure 3. Probands IgG1 anti-Tg autoantibody levels plotted against mid-parental averages assayed simultaneously (100).
show a significantly positive slope for IgG1 (Figure 3). However, only the HT/CLT EA patients and family members could be followed over time. A difference in trend was observed here, an inversion of the slope, i.e. change from a positive regression to a negative one, between the parents but only in the case of IgG4. Therefore, in the AA patients with GD, parental influences differed more than in the two EA groups, particularly when IgG4 levels were compared.
Discussion The ROMP plot technique allows one to obtain a measurable difference between the maternal and paternal contributions to heritability whether they are examined separately for different ethnic groups or on the basis of high or low IgG4 anti-Tg autoantibodies. The most striking finding in this retrospective analysis study was that half of the juvenile thyroiditis patients, (most of whom were female), reached the level of 20% IgG4 at some point of the study period. This large proportion may become an early criterion for potential IgG4-related thyroid disease. In order to determine whether this condition was hereditary, we employed ROMP, a simple preliminary genetic assay. Using ROMP, IgG4 showed preliminary evidence of heritability in both the two major autoimmune thyroid diseases, TT and CLT. Our threshold for including a thyroiditis patient in the IgG4-related disease group was an IgG4 subclass of
approximately 20% of the total antibody. This may be particularly important in non-Japanese patients in line with efforts to tailor criteria to different ethnic origins among patients with other autoimmune diseases [23–27]. Since the various research algorithms available require larger patient numbers, ROMP followed by isotype distribution pattern analysis could yield further useful insight. Although we recognize that environmental influences also exert considerable effects on the development of autoimmune thyroid disease [28–31], the measure of IgG4 may lead to predictions for selecting patients most suited to B-cell therapeutic strategies [3,32,33]. As reported previously that detectable levels of one of the two autoantibodies (anti Tg and TPO) were often present in either parent or in one or more sibs. In contrast, both autoantibodies were usually high only in the clinically affected probands. Since the responses to the two main autoantigens seem to be independent, we examined the probands with high and low IgG4 levels and that of the siblings separately. CLT patients showed a significantly positive slope in the group with high IgG4 anti-Tg titers. This suggests that autoantibody levels in the IgG4 subclass may become a marker for a heritable disease phenotype in CLT patients. In contrast, probands with low IgG4 titers gave negative heritability plots. However, IgG4 dominance can change over time and underscores the importance of repeated testing. The appearance of a positive slope may lead to the problem of the predictability of this analysis, in particular, when one or the other parent presents with a high IgG4 autoantibody titer. The number of siblings in this study was small and they had very low levels. Attempts have been made to address such problems using family-based study designs, in order to study genetic associations with large effects. However, next-generation sequencing technologies such as GWAS are extremely costly. ROMP (a cheap method) offers the advantage of not requiring parental genotypes [34,35]. Perhaps with a larger cohort of patients, such ROMP plots for age cohorts may prove feasible [36]. Previous work on the subclass distributions of autoantibodies in autoimmune thyroid disease described relative restriction to the IgG1 and IgG4 subclasses [37,38]. Perhaps this reflects a selected group of patients with high autoantibody titers or a particularly severe presentation, as seen in one of the mothers in our own study population. More varied distribution patterns among both adult and juvenile patients were observed when more sensitive assays were used [20]. Further studies on biopsies may enable the determination as to whether IgG4 elevation may be used as an early indicator of fibrotic disease. This may also facilitate the selection of family members among those screened for autoantibodies for follow-up in order to diagnose them early and initiate treatment if necessary. Perhaps early intervention could aid in preventing the progression to fibrosis [7,11], and unresponsiveness to steroid therapy, as has been shown for Mickulicz Disease patients [39]. In centers where a wider availability of family members may allow more of them to participate in studies, either at early stages of disease before treatment is initiated, a selection could be made that may
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
DOI: 10.3109/08916934.2014.897331
enable siblings to be screened over the years for early development of autoimmune diseases. This is particularly important when more than one autoantibody titer is increasing over time [40]. With respect to the siblings studies here with two autoantibodies that differ with respect to parents, the presence of more than one autoantibody could be considered as an early warning sign of autoimmune disease but not necessarily of a fibrotic variant [41–44]. In recent years several studies on autoimmune thyroid disease have been published with regard to various autoimmune diseases and ethnic groups [45–50]. Among these several are concerned with risk factors affecting susceptibility to GD/TT. An extensive comparison of susceptibility studies of both autoimmune thyroid diseases and of HT/CLT alone concludes that due to these ethnic differences, gene-gene and gene-environment interactions may be a more useful approach to study contributing or predisposing genes [17,51,52]. Renewed recognition of the role of antibodies to Tg, genetic linkage to HLA [53] and the skewed chromosomal inactivation to explain female preponderance [54,55] have aided interdisciplinary research in this area. In conclusion we suggest that screening may be able to differentiate these two subgroups of CLT patients by this simple method and allow those patients with IgG4-related thyroid disease to be treated earlier.
Declaration of interest The authors declare no conflicts of interest.
References 1. Masaki, Y., N. Kurose, and H. Umehara. 2011. IgG4-related disease: a novel lymphoproliferative disorder discovered and established in Japan in the 21st century. J. Clin. Exp. Hematop. 51: 13–20. 2. Ebbo, M., A. Grados, E. Bernit, et al. 2012. Pathologies associated with serum IgG4 elevation. Int. J. Rheumatol. 2012: 602809. 3. Mahajan, V. S., H. Mattoo, V. Deshpande, et al. 2014. IgG4-Related Disease. Annu. Rev. Pathol. Mech. Dis. 9: 315–347. 4. Li, Y., Y. Bai, and Z. Liu, et al. 2009. Immunohistochemistry of IgG4 can help subclassify Hashimoto’s autoimmune thyroiditis. Pathol. Int. 59: 636–641. 5. Zhang, J., L. Zhao, Y. Gao, et al. 2013. A classification of Hashimoto’s thyroiditis based on immunohistochemistry for IgG4 and IgG. Thyroid. 24: 364–370. 6. Deshpande, V., A. Huck, E. Ooi, et al. 2012. Fibrosing variant of Hashimoto thyroiditis is an IgG4 related disease. J. Clin. Pathol. 65: 725–728. 7. Shimizu, Y., M. Yamamoto, Y. Naishiro, et al. 2013. Necessity of early intervention for IgG4-related disease-delayed treatment induces fibrosis progression. Rheumatology (Oxford). 52: 679–683. 8. Guma, M., and G. S. Firestein. 2012. IgG4-related diseases. Best. Pract. Res. Clin. Rheumatol. 26: 425–438. 9. Palazzo, E., C. Palazzo, and M. Palazzo. 2013. IgG4-related disease. Joint Bone Spine. 81: 27–31. 10. Kamisawa, T., S. T. Chari, M. M. Lerch, et al. 2014. Recent advances in autoimmune pancreatitis: type 1 and type 2. Postgrad. Med. J. 90: 18–25. 11. Koike, T. 2013. IgG4-related disease: why high IgG4 and fibrosis? Arthritis Res. Ther. 15: 103. 12. Lee, L. H., E. A. Shaffer, V. Falck, and M. M. Kelly. 2013. Solitary lesions with fibrosis and increased IgG4+ plasma cells: Part of the expanding spectrum of IgG4-related disease or a nonspecific inflammatory response? Int. J. Surg. Pathol. [Epub ahead of print]. doi: 10.1177/1066896913513832.
IgG4-related thyroid disease heritability
325
13. Moroni, L., I. Bianchi, and A. Lleo. 2012. Geoepidemiology, gender and autoimmune disease. Autoimmun. Rev. 11: A386–92. 14. Burek, C. L., W. H. Hoffman, and N. R. Rose. 1982. The presence of thyroid autoantibodies in children and adolescents with autoimmune thyroid disease and in their siblings and parents. Clin. Immunol. Immunopathol. 25: 395–404. 15. Burek, C.L., N. R. Rose, G. M. Najar, et al. 1984. Autoimmune Thyroid Disease. In Clinical Immunology, Vol. 1 – Immunogenetics. C. S. David, and G. S. Panayi, eds. Butterworths, London. p. 207–233. 16. Li, Y., E. Nishihara, and K. Kakudo. 2011. Hashimoto’s thyroiditis: old concepts and new insights. Curr. Opin. Rheumatol. 23: 102–107. 17. Jabrocka-Hybel, A., A. Skalniak, J. Pia˛tkowski, et al. 2013. How far are we from understanding the genetic basis of Hashimoto’s thyroiditis? Int. Rev. Immunol. 32: 337–354. 18. Manolio, T. A., K. C. Barnes, T. H. Beaty, et al. 2003. Sex differences in heritability of sensitization to Blomia tropicalis in asthma using regression of offspring on midparent (ROMP) methods. Hum. Genet. 113: 437–446. 19. Outschoorn, I. M., M. V. Talor, W. H. Hoffman, et al. 2011. Autoantibody heritability in thyroiditis: IgG subclass contributions. Autoimmunity. 44: 195–200. 20. Kuppers, R. C., I. M. Outschoorn, R. G. Hamilton, et al. 1993. Quantitative measurement of human thyroglobulin-specific antibodies by use of a sensitive enzyme-linked immunoassay. Clin. Immunol. Immunopathol. 67: 68–77. 21. Beastall, G. H. 2014. What is the significance of antithyroid antibodies in children and adolescents? Clin. Biochem. 47: 1–2. 22. Taubner, K., G. Schubert, F. Pulzer, et al. 2014. Serum concentrations of anti-thyroid peroxidase and anti-thyroglobulin antibodies in children and adolescents without apparent thyroid disorders. Clin. Biochem. 47: 3–7. 23. Hart, P. A., T. Kamisawa, W. R. Brugge, et al. 2013. Long-term outcomes of autoimmune pancreatitis: a multicentre, international analysis. Gut. 62: 1771–1776. 24. Yamamoto, M., T. Tabeya, Y. Naishiro, et al. 2012. Value of serum IgG4 in the diagnosis of IgG4-related disease and in differentiation from rheumatic diseases and other diseases. Mod. Rheumatol. 22: 419–425. 25. Tsuboi, H., H. Asashima, C. Takai, et al. 2013. Primary and secondary surveys on epidemiology of Sjo¨gren’s syndrome in Japan. Mod. Rheumatol. [Epub ahead of print]. doi: 10.3109/ 14397595.2013.843765. 26. Yang, Y., X. Su, K. Zhang, and R. Zhou. 2013. The Fc receptor-like 3 gene polymorphisms and susceptibility to autoimmune diseases: an updated meta-analysis. Autoimmunity. 46: 547–558. 27. Yang, Y., K. Zhang, and R. Zhou. 2014. Meta-analysis of premiRNA polymorphisms association with susceptibility to autoimmune diseases. Immunol. Invest. 43: 13–27. 28. Duntas, L. H. 2011. Environmental factors and thyroid autoimmunity. Ann. Endocrinol. (Paris). 72: 108–113. 29. McLeod, D. S., and D. S. Cooper. 2012. The incidence and prevalence of thyroid autoimmunity. Endocrine. 42: 252–265. 30. Temajo, N. O., and N. Howard. 2014. The mosaic of environment involvement in autoimmunity: the abrogation of viral latency by stress, a non-infectious environmental agent, is an intrinsic prerequisite prelude before viruses can rank as infectious environmental agents that trigger autoimmune diseases. Autoimmun. Rev. [Epub ahead of print]. doi: 10.1016/j.autrev.2013.12.003. 31. de Buy Wenniger, L. J., E. L. Culver, and U. Beuers. 2014. Exposure to occupational antigens might predispose to IgG4related disease. Hepatology. [Epub ahead of print]. doi: 10.1002/ hep.26999. 32. Yamamoto, M., H. Takahashi, and K. Ishigami, et al. 2012. Relapse patterns in IgG4-related disease. Ann. Rheum. Dis. 71: 1755. 33. Murakami J., Matsui S., Ishizawa S., et al. 2013. Recurrence of IgG4-related disease following treatment with rituximab. Mod. Rheumatol. 23: 1226–1230. 34. Arlt, M. F., T. E. Wilson, and T. W. Glover. 2012. Replication stress and mechanisms of CNV formation. Curr. Opin. Genet. Dev. 22: 204–210.
Downloaded by [Gothenburg University Library] at 14:44 08 October 2017
326
I. M. Outschoorn et al.
35. Inoue, N., M. Watanabe, H. Yamada, et al. 2012. Associations between autoimmune thyroid disease prognosis and functional polymorphisms of susceptibility genes, CTLA4, PTPN22, CD40, FCRL3, and ZFAT, previously revealed in genome-wide association studies. J. Clin. Immunol. 32: 1243–1252. 36. Brix, T. H., and L. Hegedu¨s. 2012. Twin studies as a model for exploring the aetiology of autoimmune thyroid disease. Clin. Endocrinol (Oxf). 76: 457–464. 37. Parkes, A. B., S. M. McLachlan, P. Bird, and B. Rees Smith. 1984. The distribution of microsomal and thyroglobulin antibody activity among the IgG subclasses. Clin. Exp. Immunol. 57: 239–243. 38. McLachlan, S. M., and B. Rapoport. 2008. Thyroid peroxidase autoantibody epitopes revisited*. Clin. Endocrinol (Oxf). 69: 526–527. 39. Yamamoto, M., H. Takahashi, K. Ishigami, et al. 2012. Evaluation and clinical validity of a new questionnaire for Mikulicz’s disease. Int. J. Rheumatol. 2012: 283459. 40. Watanabe, T., M. Maruyama, T. Ito, et al. 2013. Two siblings with type 1 autoimmune pancreatitis. Intern. Med. 52: 895–899. 41. Okazaki, K., and H. Umehara. 2012. Are Classification Criteria for IgG4-RD Now Possible? The concept of IgG4-related disease and proposal of comprehensive diagnostic criteria in Japan. Int. J. Rheumatol. 2012: 357071. 42. Watanabe, T., M. Maruyama, T. Ito, et al. 2013. Clinical features of a new disease concept, IgG4-related thyroiditis. Scand. J. Rheumatol. 42: 325–330. 43. Kawashima, S. T., T. Tagami, K. Nakao, et al. 2013. Serum levels of IgG and IgG4 in Hashimoto thyroiditis. Endocrine. 45: 236–243. 44. Li, D., W. Cai, R. Gu, et al. 2013. Th17 cell plays a role in the pathogenesis of Hashimoto’s thyroiditis in patients. Clin. Immunol. 149: 411–420.
Autoimmunity, 2014; 47(5): 320–326
45. Ban, Y., T. Tozaki, M. Taniyama, et al. 2012. Multiple SNPs in intron 41 of thyroglobulin gene are associated with autoimmune thyroid disease in the Japanese population. PLoS One. 7: e37501. 46. Ban, Y., T. Tozaki, and M. Taniyama. 2013. The replication of the association of the rs9355610 within 6p27 with Graves’ disease. Autoimmunity. 46: 395–398. 47. Alkhateeb, A., N. A. Marzouka, and R. Tashtoush. 2013. Variants in PTPN22 and SMOC2 genes and the risk of thyroid disease in the Jordanian Arab population. Endocrine. 44: 702–709. 48. Alkhateeb, A., Y. Jarun, and R. Tashtoush. 2013. Polymorphisms in NLRP1 gene and susceptibility to autoimmune thyroid disease. Autoimmunity. 46: 215–221. 49. Zaaber, I., S. Mestiri, H. Marmouch, et al. 2013. Polymorphisms in TSHR and IL1RN genes and the risk and prognosis of Hashimoto’s thyroiditis. Autoimmunity. 47: 113–118. 50. Yan, N., Y. L. Yu, J. Yang, et al. 2012. Association of interleukin17A and -17F gene single-nucleotide polymorphisms with autoimmune thyroid diseases. Autoimmunity. 45: 533–539. 51. Weetman, A. 2013. A hundred years of Hashimoto’s thyroiditis. Thyroid. 23: 135–136. 52. Culver, E. L., and A. C. Bateman. 2013. General principles of IgG4-related disease. Mini-Symposium. Diagnostic Histopathology. 19:111–118. 53. Duntas, L.H., Y. Hiromatsu, and N. Amino. 2013. Centennial of the description of Hashimoto’s thyroiditis: two thought-provoking events. Thyroid. 23: 643–645. 54. Simmonds, M. J., F. K. Kavvoura, O. J. Brand, et al. 2014. Skewed x chromosome inactivation and female preponderance in autoimmune thyroid disease: an association study and meta-analysis. J. Clin. Endocrinol. Metab. 99: E127–E131. 55. Hunt, K. A., V. Mistry, N. A. Bockett, et al. 2013. Negligible impact of rare autoimmune-locus coding-region variants on missing heritability. Nature. 498: 232–235.
